SPECIFIC AMPLIFICATION OF NECATOR-AMERICANUS OR ANCYLOSTOMA-DUODENALEDNA BY PCR USING MARKERS IN ITS-1 RDNA, AND ITS IMPLICATIONS

Necator americanus and Ancylostoma duodenale are the two most importan t species of human hookworm, and occur in sympatry over much of their distribution. The specific diagnosis of hookworm infections is central to control. Diagnosis currently relies on the detection of hookworm e ggs in human faeces and/or the specific identification of larvae by 'c opro-culture' combined with microscopic examination. However, the eggs of the two species are morphologically indistinguishable, and the pro cedure of copro-culture is tedious and time-consuming to carry out. To work toward overcoming these limitations, a molecular approach utiliz ing genetic markers in the first internal transcribed spacer (ITS-1) o f ribosomal DNA (rDNA) was established. The ITS-1 sequences of both ho okworm species were determined, and specific oligonucleotide primers d esigned to regions of major sequence difference between the species we re evaluated in polymerase chain reaction (PCR). Using a range of cont rol samples, the primers allowed the specific amplification of as litt le as 10 pg DNA of A. duodenale or N. americanus. The findings indicat e clearly the potential for specific PCR to confirm the identity of eg gs from faeces and larvae from the environment or host tissues. This s hould have important implications for studying fundamental aspects rel ating to anthelmintic efficacy and the epidemiology of hookworms. (C) 1998 Academic Press Limited.